I. Field of the Invention
The invention provides a novel group of compounds for modulating cellular activities comprising sulfur-containing hydrocarbon derivatives of carboxy-amino-amides such as vitaletheine, [N-(2-mercapto-ethane)-3-carboxyamino-propanamide], also designated[N-[3-(2-mercapto-ethanamino)-3-oxo-3,1-propanediyl]carbamic acid], herein referred to as "vitaletheine modulators". The compounds of the invention are characterized by a pronounced biological activity, and are useful, inter alia, for improving the phenotypic expression and vitality of cells in culture. In particular, the compounds of the invention increase cellular lifespan, increase cellular bioproductivity, improve cellular function in culture, and adapt resistant cells to culture.
"Phenotypic cell expression" is defined herein as the manifestation of an entire range of physical, biochemical and physiological characteristics of an individual cell as determined both genetically and environmentally, in contrast to "genotypic cell expression", which in the art solely refers to the expression of the cell chromosomal sequence. [See, for example, Dorland's Illustrated Medical Dictionary, 26th Edition, 1974, W. B. Saunders, Philadelphia]. Biological activity of the vitaletheine modulators of the invention thus includes modulation of the expression of genetic material of cells in culture as influenced by the condition and environment of each cell, including the age of the cell, the culture conditions employed, and the presence of optionally added biological effectors.
II. Discussion of Related Art
Cells which are not capable of continuous growth in culture (non-immortal cells or cell lines) are characterized by a predictable lifespan in vitro, broadly divisible into three phases corresponding to growth, maturation, and decline (i.e., senescence). Cellular senescence is a phenomenon well-recognized in the art, typically characterized, inter alia, by a statistically significant lengthening of the time required for a mature individual cell to reproduce (generation time), by the elongation of normal cell growth patterns reflecting the increasing inability of the cell to efficiently incorporate essential energy and material requirements, and by the termination or statistically significant diminution of the cell's bioproductivity, which is usually optimal at midcycle (maturity). The life spans of many non-immortal cells in culture, particularly mammalian cells, frequently varies from only a matter of hours to only several weeks, even under optimal culture conditions. Sudden, premature death of such cultures is not uncommon. Even so-called immortal cells, such as immortal insect cell lines or mammalian tumor cell lines, tend to lose viability as a function of time in culture, with corresponding decline of the cell mass. Further, many cells, such as mammalian hepatic cells, cannot be presently adapted to long-term culture as a practical matter.
These inherent limitations on cell longevity in vitro have important implications for cultures employed in chemical, industrial, and research applications, and are of particular interest in the in vitro production of mammalian cell products, including recombinant cell products, especially peptides, proteins, and glycoproteins, such as hormones, enzymes, and immunoglobulins, wherein optimum production is typically obtained during the pre-senescent phases of the cell's life-cycle. A variety of methods have been proposed for maximizing the production and longevity of cells within existing limitations imposed by cell growth patterns; these are primarily directed to the improvement of culture conditions by techniques for the rapid replenishment of nutrients and removal of wastes, such as perfusion and continuous culture procedures, or to biological manipulation of cells, such as hybridization with immortalizing cell lines. While such techniques have generally tended to improve bioproductivity in large-scale applications, the improved results are not usually attributable to alteration of cell growth patterns. Further, such prior art methods for improving cell bioproductivities have not been broadly applicable to cells considered non-adaptable to culture; the hepatic cells mentioned above, for example, are currently not maintainable in vitro under known culture conditions for more than a few hours.
Methods for the biochemical modification of cell growth patterns have also been proposed to improve cell propagation, but most have been predicated on the use of cell growth factors. While growth factors as a group generally tend to increase proliferation of cells in culture, cells exposed to these factors also rapidly become exhausted and die, with little or no net gain in cell bioproductivity. Additionally, such growth factors have not been useful in adapting resistant cells to culture.
It is accordingly desirable to provide compounds which are effective for promoting the viability and propagation of cells in culture, particularly for promoting cell vitality, cell bioproductivity, cell function, and cell longevity, and for adapting resistant cells to culture. Such compounds are potentially useful not only by themselves, but also in combination with other bioeffectors which are known to promote cellular propagation, for their contemplated combined effects, such as stabilization and augmentation of the cell biomass.